Identification and functional characterization of two DNA-targeting CRISPR-Cas immune effector complexes from Pyrococcus furiosus
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There are multiple CRISPR-Cas immune systems that silence invader nucleic acids in prokaryotes via diverse effector complexes. CRISPR-Cas effector complexes are comprised of CRISPR RNAs (crRNAs) that contain short sequences captured from invaders and Cas proteins specific to the immune system type. Invader nucleic acids are recognized by crRNAs and cleaved by the complexes. The work presented here indicates that three distinct CRISPR-Cas effector complexes co-function in the hyperthermophilic archaeon Pyrococcus furiosus. We previously characterized the P. furiosus Cmr (Type III-B) effector complex, which cleaves target RNAs. Here, we have isolated two novel complexes in P. furiosus by immunoprecipitation. Tandem mass spectrometry and immunoblotting analyses revealed complexes comprised of distinct modules of Cas proteins - Csa (Type I-A) and Cst (Type I-G) as well as Cmr (Type III-B). Deep sequencing and Northern analysis revealed that processed crRNAs derived from all seven of the CRISPR loci found in P. furiosus associate with all three complexes. Furthermore, we have reconstituted active Type I-A and Type I-G effector complexes from recombinant Cas proteins and synthetic crRNA. Types I-A and Type I-G effector complexes each target and cleave non-self, double-stranded DNA (that contains a canonical Protospacer Adjacent Motif or PAM) in a crRNA-guided manner. We determined that six Csa proteins (Csa 4-1 (Cas8a2), Cas3”, Cas3’, Cas5a, Csa2 (Cas7), Csa5) and three Cst proteins (Cst1 (Cas8a2), Cas5t and Cst2 (Cas7) are each essential components of active Types I-A complexes and I-G complexes, respectively. Mutational analysis shows Cas3” and Cas3, respectively are the effector nucleases of Type I-A and I-G complexes. We have mapped the sites of cleavages mediated by Type I-A and I-G complexes and developed new models for the molecular mechanisms of DNA silencing. Finally, we delineated some features of the Csa and Cst complex architecture. Native gel shift analysis and UV-induced RNA-protein crosslinking reveal that the Cas5 and Cas7 family proteins of the Csa and Cst complexes constitute the structural core of each complex, making direct contacts with the crRNA. The 8-nt repeat-derived 5’ tag of the crRNA with 5’-OH group is critical for the assembly of Csa and Cst complexes.